PROJECT SUMMARY New Echota Biotechnology proposes to wed two highly promising technologies, CRISPR/Cas genome editing and cell-penetrating peptides (CPPs) to enhance genome editing in a wide array of cell lines, tissues and organisms. CPPs are capable of mediating penetration of the plasma membrane, allowing delivery of macromolecular cargos to the cell interior. CPP delivery of cargos to the interior compartments of cells is a potentially transformative platform technology for the development of therapeutics, diagnostics and research tools. This SBIR Phase 1 application proposes to develop an enhanced CRISPR/Cas9 product based on our novel CPP-adaptor technology. Our innovative approach to cargo delivery is the use of high affinity but reversible noncovalent binding to attach cargo to CPPs. We have designed CPP-adaptor fusion proteins consisting of the cell penetrating moiety from HIV transactivator of transcription or other CPP fused to calmodulin (CaM) or other EF hand proteins. Our prototypes bind CaM binding-site (CBS)-containing cargos with nanomolar affinity in the presence of calcium but negligibly in its absence. Because mammalian cells typically maintain low resting concentrations of calcium, cargos dissociate from the CPP-adaptor once inside the cell. The largest technical hurdle to development of CPP therapeutics is failure to escape from endosomes ? our technology solves this problem. We will deliver active Cas9/sgRNA complexes to the nuclei of mammalian cells using our proprietary CPP- adaptor technology, and using an established CRISPR assay, optimize delivery for efficiency of easily assayable editing. We will then use the optimized conditions to perform proof-of-concept experiments, knocking out F9, which encodes blood coagulation factor IX (FIX), defects in which cause hemophilia B. We will also design and execute a small mouse study to demonstrate efficacy of our approach in animals, modeling from a previous studies in which editing was effected via injection of CRISPR plasmids in tail veins. New Echota Biotechnology?s (NEB) system has speed, safety and efficiency advantages over competing methods and ease of use and dosing advantages over other CPP methods. The significance of this application is that success will enable utilization of CPP-adaptors to enhance CRISPR technology in a way that overcomes currently intractable limitations.